Properly formed heart valve leaflets are essential to achieve unidirectional blood flow and thus, are critical for life. The delicate leaflets open and close with each heart beat, promoting optimal heart function by preventing regurgitation and thus, oxygen delivery to tissues throughout the body. Congenital heart defects, many of which affect the heart valves, occur in about 1 in 100 infants. Some congenital valve defects are life threatening from the moment of birth; others are less problematic, but eventually valve replacement becomes necessary in adult life. Despite the critical role of the valves in heart function, relatively little is known about the development of the valves and even less about the cellular and molecular processes that sustain valve function through adult life. We have shown that endothelial cells from adult pulmonary and aortic valve leaflets exhibit valve specific properties that are reminiscent of critical steps in valvulogenesis, indicating that endothelial cells from adult leaflets retain the ability to recapitulate embryonic developmental processes. We have shown that in human valvular endothelial cells, activation and nuclear translocation of the transcription factor NFATc1 is required for VEGF-induced proliferation and migration, we have also shown that clonal populations of adult valvular endothelial cells can be induced to undergo an endothelial to mesenchymal transdifferentiation (EMT), a critical event in formation of valve leaflets from the endocardial cushions. The valvular endothelial cell culture models we have established provide powerful tools for identifying genes that control endothelial proliferation and differentiation and for understanding the interplay between proliferation and differentiation in cardiac valves. Three specific aims will be pursued. The first will be to identify and functionally characterize targets of the VEGF/NFATc1 signaling pathway in valve endothelium. The second will be to identify and functionally characterize genes involved in TGF-beta mediated EMT. The third aim will be to examine the cross-regulation between proliferation and differentiation in cardiac valve endothelial cells. Based on new preliminary data, we hypothesize the bone morphogenetic protein-2 (BMP-2) links these two pathways. Aim 3 has been expanded in this revised application to specifically test the role of BMP-2 in proliferation, migration and EMT in cardiac valve endothelium. The studies proposed here will provide insights on 1) normal functions and capabilities of valvular endothelium, 2) heart valve diseases and potential mechanisms for repair, and 3) strategies to create improved valve replacements for patients suffering from valve-specific defects.